Iodine was officially recognized by the Pharmacopeia of the United States in 1930, also as tincture iodine (tincture of iodine) and linimentum iodi (liniment of iodine). Clinicians and microbiologists described a great number of experimental data and clinical applications. Despite the successes that have been achieved with iodine, it was ascertained early that it also possesses properties unsuitable for practical application, including, for example, the fact that iodine has an unpleasant odor. In addition, it stains the skin with an intensive yellow-brownish color, causes blue stains in the laundry in the presence of starch, and combines with iron and other metals, its solutions are not stable, it irritates animal tissue, and is a poison. The adverse side effects of iodine, its painfulness on open wounds and the possibility of allergic reactions in the past 100 years led to the production of a great many iodine compounds (and iodine preparations), with the aim of avoiding these incompatibilities without a significant loss of germicidal efficiency. In this connection, the iodophors finally succeeded as nearly ideal forms for application of iodine.
Although exact details about the killing of a living cell by the I.sub.2 molecule (or one of the reaction products occurring in aqueous solution) are not known, it can be assumed that iodine reacts:
(1) With basic N--H functions that arc parts of some amino acids (e.g., lysine, histidine, arginine) and the bases of nucleotides (adenine, cytosine, and guanine) forming the corresponding N-iododerivatives. By this reaction, important positions for hydrogen bonding are blocked, and a lethal disorder of the protein structure may occur. PA1 (2) Oxidizing the S--H group of the amino acid cysteine, through which the connections of protein chains by disulfide (--S--S--) bridges, as an important factor in the synthesis of proteins, are lost. PA1 (3) With the phenolic group of the amino acid tyrosine, forming mono- or diiodo-derivatives. In this case, the bulk of the iodine atom(s) in the ortho position may cause a form of steric hindrance in the hydrogen bonding of the phenolic OH group. PA1 (4) With the carbon-carbon double bond (C.dbd.C) of the unsaturated fatty acids. This could lead to a change in the physical properties of the lipids and membrane immobilization. PA1 (1) A standard destruction (i.e., a 99.999% kill in 10 minutes at 25.degree. C.) of enteric bacteria, amoebic cysts, and enteric viruses requires I.sub.2 residuals of 0.2, 3.5, and 14.6 ppm, respectively. PA1 (2) On a weight basis, iodine can inactivate viruses more completely over a wide range of water quality than other halogens. PA1 (3) In the presence of organic and inorganic nitrogenous substances, iodine is the cysticide of choice because it does not produce side reactions that interfere with its disinfecting properties. PA1 (4) Iodine would require the smallest mg/L dosage compared to chlorine or bromine to "break any water" to provide a free residual. PA1 (5) I.sub.2 is 2 to 3 times as cysticidal and 6 times as sporicidal as HOI, while HOI is at least 40 times as virucidal as I.sub.2. This behavior is explained on the one hand by the higher diffusibility of molecular iodine through the cell walls of cysts and spores and on the other hand by the higher oxidizing power of HOI.
Iodine - polymer complexes, e.g., with poly(vinylpyrrolidone) (PVP), and complexes of iodine with nonionic surfactants, e.g., polyethylene glycol mono(nonylphenyl) ether have been used with considerable success. However, use in direct contact with labile biological materials has been limited because either the killing power of iodine is dissipated in the biological material or damages the biological material.
Povidone iodine is capable, in certain circumstances, of killing all classes of pathogens encountered in nosocomial infections: gram-positive and gram-negative bacteria, mycobacteria, fungi, yeasts, viruses and protozoa. Most bacteria are killed within 15 to 30 seconds of contact.
Iodine is consumed by proteinaceous substrates and its efficacy as a disinfectant is reduced at certain antiseptic applications. This is due to a reducing effect of the material to be disinfected which leads to the conversion of iodine into non-bactericidal iodide. Thus, not only the reservoir of available iodine is diminished but also the equilibrium of triiodide is influenced as well. Both of these effects cause a decrease in the proportion of free molecular iodine, the actual anti-microbial agent. When povidone-iodine preparations are contaminated with liquid substrata, e.g. blood, etc., there is, in addition, the dilution effect characteristic of povidone-iodine systems which causes an increase in the equilibrium concentration of free molecular iodine. To what extent the latter effect compensates for the other two effects depends on the content of reducing substances. Thus with full blood, a strong decrease of the concentration of free molecular iodine occurs, while, in the presence of plasma, it remains practically unchanged. Durmaz, et al, Mikrobiyol. Bul. 22 (3), 1988 (abstract); Gottardi W, Hyg. Med. 12 (4). 1987. 150-154. Nutrient broth and plasma had little inactivating activity but 1 g hemoglobin inactivated 50 mg of free I; experiments with .sup.125 I showed that uptake of I by [human] red cells occurred rapidly. Optimal antimicrobial effects in clinical use should be achieved in relatively blood-free situations. Povidone iodine produced a potent and sometimes persistent bactericidal effect towards bacteria on healthy skin. Lacey, R. W., J Appl Bacteriol 46 (3). 1979. 443-450. The bactericidal activity of dilute povidone-iodine solutions is inversely proportional to the concentration of the povidone-iodine solutions and is inhibited to the greatest extent by blood, followed by pus, fat and glove powder. Zamora J L; Surgery (St Louis) 98 (1). 1985. 25-29; Zamora, Am. J. Surgery, 151, p. 400 (1986); see also, Waheed Sheikh, Current Therapeutic Research 40, No. 6, 1096 (1986). Van Den Brock, et al, Antimicrobial Agents and Chemotherapy, 1982, 593-597, suggests that povidone-iodine is bound to cell wall proteins leaving little for interaction with microorganisms in the liquid phase (See, also, Abdullah, et al., Arzneim.Forsch./Drug Res. 31 (I), Nr. 5, 828). Ninneman et al, J. of Immunol. 81, 1265 (1981) reported that povidone-iodine was absorbed in serum albumin and it is know that povidone-iodine is bound to albumin.
Iodine is used widely in human medicine is the disinfection of skin, (e.g., the preoperative preparations of the skin, the surgical disinfection of hands, the disinfection of the perincum prior to delivery, and the disinfection of the skin prior to infections and transfusions). Iodine preparations are also used for therapeutic purposes, e.g., the treatment of infected and burned skin but is a strong irritant. Iodophors largely overcome the irritation. Iodine has also been used for the disinfection of medical equipment, such as catgut, catheters, knife blades, ampules, plastic items, rubber goods, brushes, multiple-dose vials, and thermometers. The use of iodine as an aerial disinfectant has been advocated since 1926, and experiments on the disinfection of air have been carried out, mainly during World War II. Aerial disinfection of air-raid shelters with iodine vapors as a prophylactic measure against influenza has been recommended and a "relatively tolerable" concentration of 0.1 mg/ft.sup.3 (3.5 mg/m.sup.3) was found to be sufficient for a rapid kill of freshly sprayed salivary organisms. Obviously, one is aware of the danger that iodine vapors pose to the respiratory organs, documented by the fact that the maximum allowed concentration of iodine comes to 1.0 mg/m.sup.3.
The use of "oxidizing iodine" including "compounds incorporating molecules of oxidizing iodine" e.g. absorbed or grafted on a purified vegetable carbon, as blood-contacting reagents having bactericidal and bacteriostatic action are mentioned in passing in connection with an autotransfuser device in U.S. Pat. No. 4,898,572, Surugue nee Lasnier, et al but without any explanation or elucidation.
Iodine is, thus, an excellent, prompt, effective microbicide with a broad range of action that includes almost all of the important health-related microorganisms, such as enteric bacteria, enteric viruses, bacterial viruses, and protozoan cysts, if the sometimes severe limitations inherent in its use are overcome. Mycobacteria and the spores of bacilli and clostridia can also be killed by iodine. Furthermore, iodine also exhibits a fungicidal and trichomonacidal activity. As to be expected, varying amounts of iodine are necessary to achieve complete disinfection of the different classes or organisms. Within the same class, however, the published data on the disinfecting effect of iodine correspond only to a small extent. In particular, the published killing time of spores and viruses are widely disparate.
Various authors have tried to summarize the disinfecting properties of iodine and the other halogens by reviewing the literature and analyzing the existing data. The most important conclusions are:
Gottardi, W. Iodine and Iodine Compounds in DISINFECTION, STERILIZATION, AND PRESERVATION, Third Edition, Block, Seymour S., Ed., Lea & Febigor, Philadelphia, 1983, and the references cited therein provide more details respecting the background discussed above.
Polyvinylpyrrolidone (PVP, Povidone) is manufactured by BASF Aktiengescllschaft, Unternehemensbercieh Feincheme, D-6700 Ludwigshaven, Germany and sold under the trademark KOLIDON.RTM.. Povidone-iodine products and the preparation of such products are described in U.S. Pat. Nos. 2,707,701, 2,826,532, and 2,900,305 to Hosmer and Siggia, assigned to GAF Corporation and in a number of GAF Corporation publications; see, e.g. Tableting with Povidone3/4 povidone USP (1981) and PVP Polyvinylpyrrolidone (1982).
There is extensive patent literature on the manufacture and use of various iodine-polymer complexes, exemplary of which are: U.S. Pat. No. 3,294,765, Hort, et al, 1966--manufacture of povidone-iodine complex; U.S. Pat. No. 3,468,831, Barabas, et. al., 1969--graft co-polymers of N-vinyl pyrrolidone; U.S. Pat. No. 3,468,832, Barabas, et. al., 1969--graft copolymers of N-vinyl pyrrolidone; U.S. Pat. No. 3,488,312, Barabas, et. al, 1970--water-insoluble graft polymer-iodine complexes; U.S. Pat. No. 3,689,438, Field, et. al., 1972--cross-linked polymer-iodine manufacture; U.S. Pat. No. 3,907,720, Field, et. al., 1975--cross-linked polymer-iodine manufacture; U.S. Pat. No. 4,017,407, Cantor, et. al., 1977--solid N-vinyl-2-pyrrolidone polymer carriers for iodine; U.S. Pat. No. 4,128,633, Lorenz et al, 1978--preparation of PVP-I complex; U.S. Pat. No. 4,139,688, Dixon, 1979--cross-linked vinylpyrrolidone; U.S. Pat. No. 4,180,633, Dixon, 1979--cross-linked vinylpyrrolidone; U.S. Pat. No. 4,190,718, Lorenz, et. al., 1980--increasing molecular weight of polyvinylpyrrolidone.
Under ordinary conditions, PVP is stable as a solid and in solution. The single most attractive property of PVP is its binding capability. This property has permitted utilization in numerous commercial applications. Small quantities of PVP stabilize aqueous emulsions and suspensions, apparently by its absorption as a thin layer on the surface of individual colloidal particles. The single most widely studied and best characterized PVP complex is that of PVP-iodine. For example, hydrogen triiodide forms a complex with PVP that is so stable that there is no appreciable vapor pressure. It is superior to tincture of iodine as a germicide.
Various poloxamers (i.e., polyether alcohols) also make effective carriers/or iodine (i.e., Prepodyne, Septodyne) that exhibit the same germicidal activity as povidone-iodine. The iodophors are available in a variety of forms, such as a 10% applicator solution, 2% cleansing solution (scrub), aerosol spray, aerosol foam, vaginal gel (for trichonomal and candidal infections) ointment powder, mouthwash, perineal wash, and whirlpool concentrate (all 2%). All iodophors may be used in this invention in some of its various uses and applications and, to the extent that the iodophor is effective and does not injure the material undergoing treatment, are considered generally as equivalents or potential equivalents of povidone iodine.
As used here, the term "blood" means whole blood and blood fractions, components, and products of blood, unless "whole blood" or a specific blood derivative, e.g. a blood fraction, component or product of blood is stated. Thus, the term "blood" may apply to whole blood at the time of collection or a blood derivative at any stage in processing, as indicated by context. Blood derivatives mean blood components such as blood cell concentrates (red blood cells, platelets, etc.), plasma, and serum and products and factors prepared from blood such as albumin and the blood factors. Body tissues and cells means any tissue(s), organ(s) or cells or fluids which contain tissue(s), organ(s) or cells of animal origin. Thus, in a broad sense, body tissues and cells include blood and the cellular components of blood; however, for the most part, simply for clarity in presentation, blood is treated as a separate application of the invention. Examples of body tissues and cells include sperm, bone marrow, kidneys, cornea, heart valves, tendons, ligaments, skin, homograft or xenograft implants and prosthesis generally. Tissue and cell cultures means cells and tissues grown or enhanced in culture media and the culture media per se, but not including nutrients intended for use in cell cultures. Examples of a cultured tissue is cultured skin tissue for use in burn victims, cells and cellular products prepared by standard biological and/or genetic engineering techniques arc other examples of tissue cultures. Laboratory reagents and standards, as used in this specification and the claims, means reagents and standards produced from or comprising human or animal fluids, cells or tissues. Examples of such products are red blood cell panel utilized for typing blood, control sera and chemistry controls. Samples of tissues and fluids to be tested include samples of blood, urine, sputum, cell smears, etc. While the term "donor" is not usually applied to the individual from whom such samples are acquired, that term, "donor" will be used here in a more general sense to include the individual from whom any blood, tissue, cells or fluid is obtained for any purpose, and such term will be used to refer even to an unwilling donor.
If a tissue is explanted into the culture media for the purpose of propagating its cells, the procedure is called tissue culture whereas the explanting of individual cells into culture media would be called cell culture; however, both procedures are often referred to by the term "tissue culture" procedures without differentiation, unless the distinction is critical for some ancillary reason. This general usage of the term is employed here.
Tissue cultured cells are extremely fragile in many ways, having exacting requirements not only as to nutrients but also to the amount and type of resident organisms which can be tolerated, and culture media are highly susceptible to bacterial and/or viral infection.
Povidone is used generally to describe compounds described in the U.S. Pharmacopeia to identify polyvinyl pyrrolidone suitable for use in physiologically acceptable solutions and to include polyvinylpyrrolidone (PVP) compositions that have not yet been approved for use in the preparation of therapeutic compounds, and equivalents, as described hereinbefore. When percent concentrations are referred to in connection with povidone-iodine, the percentage refers to the percent of povidone-iodine by weight, based upon the weight of the solution or material to which the povidone-iodine is added. Thus, a 1 weight percent (w/o) solution of povidone-iodine indicates that enough povidone-iodine has been dissolved to result in a concentration of 1 w/o povidone-iodine. The ratio of polyvinyl pyrrolidone to iodine in the povidone-iodine product used in the experiments referred to hereinafter is 8.5 parts of povidone-iodine per 1 part of active iodine. The product also contains about 0.5 parts of inactive iodine as iodide. Typical stock solutions are 10% (10,000 ppm I.sub.2), 5% (5,000 ppm I.sub.2) and 1% (100 ppm I.sub.2). In those instances in which a povidone to iodine ratio of higher than about 8.5 to 1 is referred to, additional povidone (polyvinyl pyrrolidone) is added to increase the PVP to I.sub.2 ratio. The concentration of povidone-iodine in such compositions means the concentration of standard PVP-I (calculated as having an 8:5 to 1 PVP to I.sub.2 ratio, whether or not added in that ratio. PVP in excess is treated, for purposes of calculation, separately from the PVP in "standard" povidone iodine.
PVP-I-PVP is used as an abbreviation for povidone enriched povidone iodine, i.e. a composition in which the total povidone to iodine ratio is greater than 15 to 1.
PVP-I-PVPLMW is used as an abbreviation for PVP-I-PVP in which at least ten percent of the povidone has a molecular weight of no greater than approximately 12,000 daltons.
PVP-I-PVPXL is used as an abbreviation for a composition that, as to synthetic polymeric materials, consists essentially of soluble PVP-I and solid PVP, typically cross-linked PVP.
XLPVP-I is used as an abbreviation for iodinated solid, e.g. cross-linked, povidone.
Those who deal with blood and other invasively obtained body fluid samples risk infection from the samples. Those at risk include the doctor, nurse or clinical technician who takes the sample, the technicians who handle the sample and who use the sample in conducting analyses and tests, those who handle the sampling and testing equipment and apparatus, and the entire chain of individuals who attend to the disposal of sampling apparatus and the like, from the individuals who pick up the used apparatus through those who ultimately dispose of the apparatus, usually in specially designed high temperature furnaces. The risk is substantial, as evidenced by the fact that nearly all health care professionals with long experience carry the Epstein-Barr virus (EBV) and/or cytomegalovirus (CMV). Other pathogenic viruses to which health care workers, and those who handle blood and fluid sampling and handling apparatus, are exposed include hepatitis and human immunodeficiency virus (HIV) as well as a large number of less life-threatening viruses.
Another organism which is frequently present in blood and blood products or tractions and which presents a serious risk in certain procedures is the bacteria Yersinia enterocolitica which is become a serious contaminant, surpassing Salmonella and Campylobacter as a cause of acute bacterial gastroenteritis. A significant increase in transfusion related infections of Y. enterocolitica has been reported, Tipple, et al., Transfusion 30, 3, p.207 (1990). Y. enterocolitica and other bacteria which propagate at relatively low temperatures, e.g. Staphylococcus epidermidis and Legionella pneumophila, present, potentially, a serious threat in blood products.
In addition to the risk of transmitting infectious disease via blood or blood products, the growth of bacteria in blood and blood products at various stages of production and processing introduces pyrogens into the blood component or product which must be removed before the product can be used in therapy. Introduction of molecular iodine, e.g. povidone-I.sub.2, at an early stage in processing of blood products greatly reduces or eliminates the pyrogen-load of the ultimate product or fraction.
Protozoa give rise to many diseases, some of great medical and economic importance. Examples of such protozoa are the genus Plasmodium, e.g. P. falciparum, P. malariae, P. ovale and P. vivax, which causes malaria, Trypanosoma, which causes Chagas' disease, and Leishmania, which cause a variety of leishmaniasis. The method of this invention is effective in eliminating these causative organisms in blood and blood products.
Many viruses, in both animals and humans, may be transmitted by artificial insemination using sperm from infected individuals. Bovine leucosis (Mateva, V. et al, Monatsh. Veterinaermed. 1987, 42(9) 310) and bovine rhinotracheitis virus arc transmitted by sperm of infected bulls. (Kupferschmied, H. U., et al Theriogenology 1986, 25(3) 439). Singh, E. L. ((10th Int. Cong. on Animal Repr. and Artificial Insemination, Cong. Proc. V.I-IV, 1984) concluded that some viruses, e.g. bluetongue virus (BTV), infectious bovine rhinotracheitis virus (IBRV), bovine viral diarrhea virus (BVDV), foot and mouth virus (FMDV), akabane virus (AV) and bovine parvovirus (BPVP), were transmitted via seminal fluid rather than in the sperm cell.
Generally, this invention is applicable to the treatment of donated blood and products produced from blood, tissues and fluids for inactivating virus, bacteria, chlamydia, rickettsia, mycoplasma and other potentially pathogenic microorganisms.
Among the important potential pathogens to which this invention is applicable is cytomegalovirus (CMV). Herpesviruses, of which CMV is a member, represent a very large group of viruses which are responsible for, or involved in, cold sores, shingles, a venereal disease, mononucleosis, eye infections, birth detects and probably several cancers. The present invention is also useful in preventing the transmission of human immunodeficiency virus (HIV). While testing has made blood products safer than it was a decade ago, the complete elimination of HIV contaminated blood and blood products has not been possible using present knowledge and technology.
Blood plasma is used in the production of many important blood fractions, components and products. Transfusion plasma, per se, is frequently prepared as a single blood bag product; however, many plasma fractions and products are produced from large pools of plasma. There is a real and serious risk of infection to the technicians who handle individual blood bags and serum bags, and the risk of infection is multiplied many times in the handling of pooled plasma. There is, of course, a serious risk that the recipient of plasma or a plasma traction or product may be infected unless suitable steps are taken to kill or inactivate potentially pathogenic organisms. Such steps are usually taken far down the chain of processing steps and frequently as the final step before use, storage or lyophilization, according to the product.
The production of pyrogens in plasma and plasma products during initial handling or handling down-stream in the process chain by the propagation of organisms which, at a later stage in processing, are inactivated or killed constitutes a serious problem to producers of plasma fractions and products. Pyrogen production could be eliminated or substantially reduced if pyrogen producing organisms were killed early in the process, e.g. in the initial whole blood or in the pooled plasma.
Virus infections, among the most serious being hepatitis, present a constant and serious risk to both handlers and recipients of blood and blood products. It has been shown that fractionation workers, particularly those engaged in the preparation of plasma pools, arc at high risk of developing hepatitis B. The high risk products are fibrinogen, AHF, and prothrombin complex. The low risk products arc ISG, PPF, and albumin. The lack of infectivity of PPF and albumin is attributable to heating the final products at 60.degree. C. for 10 hours; however, such process steps tend to denature certain products and are unsuitable in the preparation of heat sensitive products.
The risks of infection from whole blood are well-known. One of the great tragedies of modern medicine is the infection of many patients, most frequently hemophiliacs who require frequent blood transfusions, with HIV. The purification of the nation's and the world's whole blood for transfusion would constitute a monumental step forward in the history of medicine. The risks of infection from red blood cell concentrates is similar to comparable risks associated with whole blood.
The teachings of the prior art suggest that neither elemental (diatomic) iodine nor complexed iodine, e.g. PVP-I.sub.2, would be an effective and reliable biocide in a fluid or in a body, e.g. blood, packed or concentrated cells, organs, etc. in which massive amounts of protein are be available to react with the iodine.
The use of povidone-iodine as a spermicide is known and one would not consider povidone-iodine as a candidate for killing pathogenic microbes in sperm-carrying liquids.
Various medical and blood handling procedures are referred to hereinafter. These are all well-known procedures and steps in these procedures are fully described in the literature. The following references are provided for general background and as sources for detailed reference to the literature as to specific procedures: TECHNICAL MANUAL of the American Association of Blood Bankers, 9th Ed. (1985); HLA TECHNIQUES FOR BLOOD BANKERS, American Association of Blood Bankers (1984); Developments in Biological Standardization, Vols. 1-57, S. Karger, Basel; CLINICAL IMMUNOCHEMISTRY, The American Association for Clinical Chemistry; MEDICINE, Vols. 1-2, Scientific American, New York; Care of the SURGICAL PATIENT, Vols 1-2, Scientific American, New York; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley-Interscience, John Wiley & Sons, New York.